We propose to create sufficient porosity in thin tissue specimens by controlling the eutectic freezing rate in lyophilization so that in conjunction with proprietary microporous film technology, we can change the means for moving reactants into and through dense tissue matrices from the current static diffusion format to a dynamic perfusion format. Our objective is to increase the efficiency of cell analysis in situ (i.e., decrease time required to achieve a 10:1 sensitivity from hours to minutes and reduce reactant volumes to multiples of tissue void volumes) and thereby cut assay cost and streamline automation. Phase 1 results validated composite film technology needed to perfuse specimens affixed to them and showed that lyophilization could be controlled to create sufficient porosity in 6um thick cryostat breast cancer specimens to perfuse aqueous reagents at a rate of 2ml/min/cm2 at 10psi, without compromising the defining parameters of cell analysis in situ (i.e., cytomorphology, signal precision and assay sensitivity). Achievement of this "Perfusion Porosity" increased sensitivity of Her-2/neu and progesterone receptor immunoassays more than 10-fold, compared to using non-porous, conventionally fixed specimens thaw-mounted on glass microscope slides. Perfusion cut the time needed to wash non-specific antibodies from specimens from 15 minutes to 1 minute, and reagent volumes from 750ml to 2ml without dissociating specifically bound antibody. We now propose to optimize specimen, lyophilization and perfusion variables to develop perfusion technology for in situ hybridization and immunocytochemistry, systematically adapting each assay step (blocking, probe incubations and washing) into a cycle that reduces total assay time to minutes, utilizing reagent volumes which are based on tissue void volumes. Prototypes for three Phase 3 products will be developed: 1) Oncyte composite thin films; 2)an inexpensive freeze-dryer dedicated to creating Perfusion Porosity in thin flat specimens; and 3)a manifold to perfuse multiple specimens at once. PROPOSED COMMERCIAL APPLICATION: Perfusion-based cell analysis will, by increasing assay efficiency, cut assay cost: By virtue of a parallel effect on assay sensitivity, the technology will become the industry standard, hasten the discovery of new markers and the evolution to clinical practice, and add value to our automated design concept.